WO1995008624A1 - PHOSPHOLIPASE C-α HUMAINE ET SEQUENCE D'ADN CODANT POUR CET ENZYME - Google Patents

PHOSPHOLIPASE C-α HUMAINE ET SEQUENCE D'ADN CODANT POUR CET ENZYME Download PDF

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Publication number
WO1995008624A1
WO1995008624A1 PCT/JP1994/001572 JP9401572W WO9508624A1 WO 1995008624 A1 WO1995008624 A1 WO 1995008624A1 JP 9401572 W JP9401572 W JP 9401572W WO 9508624 A1 WO9508624 A1 WO 9508624A1
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Prior art keywords
plc
lys
leu
ala
glu
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PCT/JP1994/001572
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English (en)
Japanese (ja)
Inventor
Naoto Hirano
Hisamaru Hirai
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Shionogi & Co., Ltd.
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Priority to EP94927098A priority Critical patent/EP0731164A4/fr
Priority to US08/627,907 priority patent/US6060302A/en
Publication of WO1995008624A1 publication Critical patent/WO1995008624A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a human phospholipase C- ⁇ , which is a secretory protein having redox activity, and a gene encoding said human phospholipase C- ⁇ .
  • the present invention relates to an expression vector containing the gene and a transformant having the expression vector.
  • PLC- ⁇ host lipase C- a
  • PLC- ⁇ host lipase C- a
  • This PLC is an enzyme that hydrolyzes glyceline and sphingolin lipids (hereinafter referred to as PLC activity), and is present in the spleen, small intestinal mucosa, and placenta of mammals. It is known that it plays an important role in vivo.
  • PLC activity hydrolyzes glyceline and sphingolin lipids
  • PI-PLC phosphatidylinositol C
  • Hydrolysis of phosphoric acid produces 1,2-diacylglycerol and inositol 4,5-triphosphoric acid (Rhee.
  • PLC-a The function of PLC-a is not well understood, but its expression is known to increase in stressed organisms and organisms with cancerous tissues.
  • Examples of mammalian PLC- ⁇ include mouse (Herapel. WM et al., J. Immunol. 146, 3713-3720 (1991)) and rat (Bennett, CF. et al., Nature 334.268). -270 (1988)) and PLC (Hirano et al., Proceedings of the 15th Annual Meeting of the Molecular Biology 4L-23, 1992) have reported PLC- ⁇ . These PLC-a have no significant homology to any of the known sequences of other known PLC family members.
  • the mammalian PLC- ⁇ described above has two conserved amino acid sequences, 1 ⁇ -Cys-Gly-His-Cys-Lys, at two sites.
  • This amino acid-preserved sequence is identical to that of amino acids, protein disulfide isomerase (PDI) and thiore doxin. It is identical or very similar to the amino acid sequence of the active site of redox activity.
  • PDI and thioredoxin are both protein disulphide reductases and isomerases. It is a multifunctional protein that acts by catalysis and catalyzes a conversion reaction between a thiol group and a disulfide group.
  • Mammalian PDI has two conserved sequences in the amino acid sequence, the PLC- ⁇ and the conserved sequence, and prokaryotic and eukaryotic thioredoxins contain The amino acid sequence has one Trp-Cys-Gly-Pro-Cys-Lys similar to the conserved sequence described above.
  • the Cys residues in these sequences are presumed to be catalytically active sites (Holmgren. AL Biol. Chei. 264.13963-13966, Freedman, RB Cell 57.1 069-1702) o
  • the present inventors have proposed that the expression of E. coli
  • PLC- ⁇ is a PLC superfamily. It belongs to a family of redox-regulated proteins that does not belong to one family and has been renamed thymuredoxin (Hirano et al., Supra). This is supported by the commonality of column E above.
  • PLC- ⁇ is a gene transformed with the oncogene v-src gene. Expression level is elevated in target cells, which may be related to carcinogenesis (Hirai. H et al. Genes Dev 4.2342-2352 (1990), Moj. Cell. Biol.10.1307-1318 (1990), Proc Natl. Acad. S ci. USA 87.8592-8596 (1990)) o
  • Human thiodoxin is a factor derived from adult T-cell leukemia (ATL).
  • ATL-derived factor also known as (ATL-derived factor), it is known to induce the expression of interlokin-12 receptor and stimulate cell proliferation (Tagaya, Y. et al., EMB0 J. 8, 757-764 (1989)) 0 Protein levels of thioredoxin are generally high in actively proliferating tissues and elevated levels of thioredoxin raRNA. (Jones, SW et al., J. Biol. Chem.
  • An object of the present invention is to solve the above-mentioned conventional problems, and an object of the present invention is to provide a human PLC-a, a human PLC-like gene, an expression vector containing the gene, and a transformation having the expression vector. In providing the body.
  • the present inventors have obtained a cypress csk protein (Noda, S. et al., 351, 69-72 (1991)) from the thymus and partially purified the cyst csk protein. A linear antiserum was prepared. Using this antiserum, the thymus cDNA library was screened to find the DNAS2 sequence encoding the oxy PLC-gene. Furthermore, isolation of the human PLC-cDNA from the human placenta cDNA library by the hybridization method using this PLC-fr cDNA. As a result, the present invention has been completed.
  • polypeptide of the present invention is a Ser at position 1 in SEQ ID NO: 1 in the sequence listing. It contains the amino acid sequence from position 481 to Leu.
  • the DNA sequence of the present invention encodes an amino acid sequence from Ser at position 1 to Leu at position 481 in SEQ ID NO: 1 in the sequence listing.
  • the expression vector of the present invention has the above DNA sequence.
  • the transformant of the present invention can be obtained by introducing the above-mentioned expression vector into a host.
  • FIG. 1 shows the amino acid sequences of PLC-like genes of mouse, human, and mouse.
  • FIG. 2 shows the amino acid sequences of human PLC- and human PDI genes.
  • FIG. 3 shows the amino acid sequences of human PLC- ⁇ and human thioredoxin arresters.
  • FIG. 4 is a diagram showing the results of measuring PLC activity in a crude extract, cytoplasmic fraction and membrane fraction of animal cells transformed with the Pseudo PLC- ⁇ gene.
  • - Figure 5 shows the measurement of the insulin-degrading activity of native and mutated PLC-a proteins produced by Escherichia coli transformed with native and mutated PLC- ⁇ genes. It is a figure showing a result.
  • polypeptide of the present invention and the DNA sequence encoding the same are prepared, for example, according to the following steps.
  • the human placenta cDNA library is prepared by a conventional method. First, all BNAs are separated from the human placenta by a method such as the Guanidine.Funol / Cross-mouth Holm method (Gene.28 (1984) .263-270). The polyclonal RNA is obtained by repeatedly subjecting the DNA to an oligo dT cellulose chromatograph to obtain polyA RNA, and a double-stranded cDNA is synthesized from the BNA. Synthesis of the cDNA can be performed according to the method of Gubler and Hofrafra (Gene, 25 (1983) .263-269).
  • a human placenta cDNA library is prepared.
  • a cloning vector a phage vector such as gtll is preferably used, but is not limited thereto, and a cloning vector known to those skilled in the art. Either cutter may be used.
  • Drosophila csk protein can be partially purified from Drosophila thymus according to a protein purification method known to those skilled in the art, for example, the method of Noda et al.
  • Polyclonal antiserum is obtained by a method known to those skilled in the art, for example, after immunizing a rabbit with a partially purified porcine csk protein, collecting the serum and precipitating it by ammonium sulfate precipitation. It can be prepared by purification. Instead of partially purified ⁇ csl (protein, a synthesized ⁇ csk protein or a fragment thereof may be used.
  • the thymus thymus cDNA library can be prepared using the thymus thymus as a material by a conventional method in the same manner as in (1) Preparation of the human placenta cDNA library.
  • the plaque derived from the phage into which the thymus cDNA was inserted for example, a ProtoBlot system (promega) was used. And screening with the polyclonal antiserum obtained in (ii) above, whereby the PLC-a protein DNA fragment can be excised. Positive phage clones can be isolated.
  • the sequencing of the DNA fragment of the positive phage clone obtained in the above (iv) can be directly performed using a method known to those skilled in the art, for example, a DNA sequencing kit. .
  • sequencing can be performed using phage DNA containing the DNA insert prepared from the clone. Preparation of the phage DNA containing the above DNA insert fragment was performed according to the protocol of Ie *, Maniatis et al., Molecular Cloning. A Laboratory manual, Cold Spring Harbor Laboratory Press, New York (1982). You can do this with The thus determined DNA sequence of thymus cDNA clone and the corresponding amino acid sequence are shown in SEQ ID NO: 2 in the Sequence Listing. (3) Isolation of human thymus cDNA phage clone
  • the human placental cDNA library obtained in the above (1) was probed with 32 P-labeled mouse PLC- ⁇ cDNA under mild hybridization conditions. Then, positive phage clones containing human PLC-DNA fragments can be isolated.
  • the probe of the above-mentioned 32 P-labeled mouse PLC- ⁇ cDNA can be prepared by a method known to those skilled in the art based on the sequence E determined in the above (V).
  • the DNA insert fragment base sequence of the positive phage clone obtained in the above section (3) can be determined by the same method as in the above (V).
  • the DNA sequence of the determined human thymus cDNA clone and the corresponding amino acid sequence are shown in SEQ ID NO: 1 in the sequence listing.
  • the human PLC- ⁇ protein can be produced using a gene recombination technique using a host ⁇ expression vector known to those skilled in the art. Many different host / expression vector combinations can be used for expression of the human PLC- ⁇ protein of the present invention.
  • useful expression vectors can include chromosomal, non-chromosomal, and synthetic DNA sequence segments.
  • Preferred examples are SV40 and various known derivatives of known bacterial plasmids. Plasmids from E.
  • coli such as pCRl, pBR322, pMB9, pET-3A, and their derivatives, the broader host range of the plasmid RP4, many derivatives of ⁇ phage, 13 And phage DNA such as single-filament phage DNA, 2 Yeast plasmids such as plasmids or derivatives thereof, plasmids containing the adenovirus major late promoter enhanced by the presence of the SV40 enhancer. And so on.
  • expression control sequences i.e., sequences that regulate expression of a DNA sequence when operably linked thereto, are described above in order to express the human PLC- ⁇ protein of the present invention.
  • useful expression control sequences include, for example, the early and late promoters of SV40, the early promoter of adenovirus or site megalovirus, the lac series, Trp system, TAC or TRC system, T7 promoter whose expression is induced by T7 RNA polymerase, major operator region and ⁇ phage Motor domain, regulatory domain of fd coat protein, promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, acidic phosphoproteinase, e.g.
  • Pho5 promoter Promoters for yeast ⁇ -mating factor polynuclear promoters in the Pacu-Virus virus system and known regulation of the expression of prokaryotic or eukaryotic cells or genes of these viruses Other ⁇ columns, and their various It encompasses combinations.
  • Single-cell host cells are useful for expressing the human PLC- ⁇ protein of the present invention.
  • These hosts include Escherichia coli, Pseudomonas, genus, * Bacillus, Streptomyces, and Saccharomyc es) and other strains of bacteria, chiney shamster ovary ("CH0") cells and cultured mouse cells, C0S1, C0S7, BSC1, BSC40 and And eukaryotic and prokaryotic cells such as animal cells, cultured insect cells, cultured human cells, and cultured plant cells such as African and BMT10 African monkey cells.
  • CH0 chiney shamster ovary
  • C0S1, C0S7, BSC1, BSC40 chiney shamster ovary
  • eukaryotic and prokaryotic cells such as animal cells, cultured insect cells, cultured human cells, and cultured plant cells such as African and BMT10 African monkey cells.
  • a vector having the DNA sequence of the human PLC- ⁇ polypeptide is introduced into a host to obtain a transformant. By culturing this in an appropriate medium, human PLC- ⁇ polypeptide is produced.
  • human PLC-a a secreted protein having redox activity, human PLC-a arrested gene, an expression vector containing the gene, and an expression vector containing the gene A transformant having a protein is provided.
  • polypeptides of the present invention have a conserved sequence that is identical or very similar to the active site of thioredoxin, which stimulates cell growth, and has a protein-like disant. Since it has activity to reduce sulfide bonds, it can be used as an anti-inflammatory agent. In addition, since the expression of PLC- ⁇ increases in vivo at the time of canceration or stress, it can be used as an index of these symptoms, and can also provide a measurement system for clinical evaluation of these symptoms.
  • the first and second strands of cDNA were synthesized from polyA RNA prepared from human placenta, and EcoRI-Notl-BamH was added to both ends of the resulting double-stranded DNA.
  • First and second strands of cDNA were synthesized from polyA RNA prepared from thymus gland, and EcoRI linkers were added to both ends of the obtained DNA. It was inserted into the EcoRI site to obtain a cDNA library.
  • the EcoRI-EcoRV fragment of the phage clone ⁇ 12 insert (located closest to the 5 ′ end of the phage clone ⁇ 12 insert) was used as the probe and The library was screened to obtain phage clone ⁇ 21 having a DNA insert longer than the phage clone ⁇ 12 insertion fragment.
  • the sequence of the inserted fragment in the obtained phage clone ⁇ 21 is as described above.
  • the phage clone; 121 contained the full length PLC- ⁇ sequence containing the sequence of the 5 ′ portion described above.
  • the full-length DNA sequence of the thus-obtained PLC-a and the corresponding amino acid sequence obtained in this manner are shown in E sequence No. 2 of the sequence listing.
  • the ⁇ ZAP11 human placenta cDNA library obtained in (1) above was subjected to mild high pre-digestion conditions. Then, the inserted DNA fragment of phage clone ⁇ 21 labeled with 32 P was screened as a probe to obtain a positive phage clone designated as ⁇ ⁇ 7. Was.
  • the sequence of the inserted fragment in the obtained phage clone was determined by the same method as in the above (1) ( ⁇ ).
  • the obtained human PLC-like full-length DNA system!] And the corresponding amino acid sequence are shown in SEQ ID NO: 1 in the sequence listing.
  • FIG. 1 shows the determined amino acid sequence of the human and human PLC- ⁇ and the amino acid sequence of the known mouse PLC- ⁇ (Hempel et al., Supra).
  • Each of the obtained mouse and human PLC-a cDNA sequences consisted of an open reading frame of 1515 bp and contained a hydrophobic N-terminal signal peptide (amino acid residue). It encodes a 505 amino acid polypeptide having a group -24 to -1). The molecular weight of each polypeptide was calculated to be 56.895 and 56,698 Da.
  • the amino acid @ ⁇ sequence of " ⁇ PLC-" showed 903 ⁇ 4, 87%. 943 ⁇ 4 homology, respectively, compared to the mouse, rat and human PLC- C sequences. .
  • Figure 2 shows the sequence of the determined human PLC- ⁇ and the known human tan.
  • 1 shows the sequence of protein disulphide reductase (PDI) (Pihlajaniemi, T. et al., EMBQ J. 6, 643-649 (1987)).
  • Human PLC-na had a homozygosity of 563 ⁇ 4 and a similarity of 32S at the amino acid level compared to human PDI.
  • FIG. 3 shows the determined sequence of human PLC-like column E and known human thioredoxin.
  • the amino acid at positions 1-105 of the human PLC-sequence is 55% smaller than that of human thioredoxin (Tagaya, Y. et al., EMBQ J. 8.75 7-764 (1989)). It showed homology and 23% similarity.
  • the PLC- ⁇ cDNAE sequence obtained in Example 1 was inserted in the forward direction of the mammalian cell expression vector pUC-CAGGS; downstream of the 3 actin promoter. And an antisense vector in which the above-mentioned PLC-a cDNA sequence was inserted in the reverse direction.
  • NIH3T3 mouse fibroblasts were transformed together with pSV2NE0 by the calcium phosphate co-precipitation method known to those skilled in the art. Changed.
  • the resulting transformants were selected with 80Q / zg / inl G418, and three independent and stable transfectants with high expression of Pseudo PLC-a protein into which the vector was introduced were introduced. (Independently named F01, F02s F03) and two independent transformants (named R01, R06) into which the antisense vector was introduced.
  • the cells of the obtained transformants F01, F02, F03, RO1 and R06 were collected and 50 mM Tris-HCl pH 7.4, 150 mM NaCl. 0.05% (W / V) Sodium sulfate, l3 ⁇ 4 (v / v) Triton X-100, lOU / nil abrotinin, 2raM PMSF, lOOmg / 1 loliptin, and lmM orthoton, '
  • the cells were lysed on ice in a cell lysate containing sodium nadiruna.
  • the obtained lysate was separated by SDS-PAGE using 93 ⁇ 4 acryloamide, and transferred to a polyvinylidene difluoride membrane (Mi 11 pore). Then, using a polyclonal antiserum 507, immuno-prototyping was carried out to measure the expression level of PLC in three cell lines.
  • the PLC-expression levels of the three stable transformants were 10 to 15 times that of endogenous PLC- ⁇ .
  • a 1.8 kb fragment of phage clone ⁇ 12 obtained in Example 1 was treated with Mung bean nucl ease to make both ends blunt.
  • the obtained fragment was subcloned into the Smal site of the bacterial expression vector pGEX2T (Pharraacia) to construct a plasmid pGEX-PLC-.
  • This plasmid was used to transform E. coli -1B.
  • the obtained transformant was cultured overnight according to a conventional method, and the obtained culture was diluted 1:10 with a new medium.
  • Trp-Cys-Gly-His-Cys-Lys was site-specifically mutated to prepare a mutant PLC-ff DNAE sequence.
  • the site-specific mutation was performed using Muta-Gene kit (Bio-Bad). Mutation primers 5 '-GCCCCCTGGTCTCCACACAGCAAAAAGCTT-3' (sense) and 5 ⁇ -GCTCCTTGGTCTGGTCACTCTAAGAATCTG-3 '(sense) have Cys at positions 33 and 36 as Ser, and Cys at positions 382 and 385. s was changed to Ser. The obtained site-specific mutation was confirmed by determining the nucleotide sequence.
  • the mutant PLC-protein replaced four Cys residues with Ser in both conserved sequences. That is, this mutant PLC-ff protein has two Trp-Ser-Gly-His-Ser-Lys sequences.
  • the mutant PLC-ct DNA sequence was expressed in Escherichia coli in the same manner as the above-mentioned natural PLC-a DNA sequence to obtain a mutant PLC-protein.
  • the insulin digestion assay was performed in the same manner as Holgeman et al. (Supra) except that the reaction volume was 60 // 1. Non-enzymatic degradation of insulin by dithiothreitol recorded as control did.
  • Escherichia coli thioredoxin was purchased from Promega.
  • Figure 5 shows the results of the measurement using the standard Atsushi. As shown by the curve connecting the black circles and the white squares in the figure, the natural type PLC-flt showed insulin reducing action, but as shown by the curves connecting the white squares. Mutant strain PLC- ⁇ did not show any insulin reducing action.
  • thioredoxin which is represented by a curve connecting white and black squares, has two to three times the activity per mole of protein than tandem PLC-. Had.
  • PLC- ⁇ has the ability to catalyze the reduction of disulfide bonds, similar to thioredoxin, and exhibits Trp-Cys-Gly-Hi The two Cys residues in it were found to be essential for its redox activity.
  • the F02 and R06 transformant cells were grown in a 150 mra culture dish containing DMEM medium (GIBC0) supplemented with 10 ⁇ l serum. The grown cells were washed with a phosphate buffer and cultured in 20 ml of a serum-free medium (Cell Grosser P. Sumitomo Pharmamaceuticals Co.). After 10 hours, collect the culture medium4. The mixture was centrifuged at 100.000 g for 1 hour at C, and the supernatant was collected. The obtained supernatant was dialyzed against water and freeze-dried.
  • DMEM medium GIBC0
  • serum-free medium Cell Grosser P. Sumitomo Pharmamaceuticals Co.
  • the F02 and E06 transformant cells grown on the culture dish in the same manner as described above were washed with DMEM medium containing no methionine, and 15 ml of a medium containing 1 ml of dialyzed sera.
  • the cells were in vivo labeled with linCi [, ">> S] Met in DMEM medium without thionin at 37.
  • the culture supernatant of the in vivo-labeled transformant cells was used as in Example 4.
  • the obtained culture supernatant was subjected to 9XSDS-PAGE, and then subjected to Fuji Bass imaging plate. £ (Fuji) to detect secreted PLC- ⁇ . In addition, it was confirmed that PLC- was highly secreted in the culture supernatant.
  • Sequence type nucleic acid
  • GGCGCCGACC TCCGCAGTCC CAGCCGAGCC
  • GCGACCCTTC CGGCCGTCCC CACCCCACCT 60 CGCCGCC ATG CGC CTC CGC CGC CTA GCG CTG TTC CCG GGT GTG GCG CTG 109
  • GCA AAG AAA TTC CTG GAT GCT GGG CAC AAA CTC AAC TTT GCT GTA GCT 973 Ala Lys Lys Phe Leu Asp Ala Gly His Lys Leu Asn Phe Ala Val Ala
  • ACTTTGTAAA AGGACTCTTC CATCAGAGAT GGAAAACCAT TGGGGAGGGA CTAGGACCCA 1662 TATGGGAATT ATTACCTCTC AGGGCCGAGA GGACAGAATG GAAATAATCT GAATCCTGTT 1722 AAATTTTCTC TAAACTGTTT CTTAGCTGCA CTGTTTATGG AAATACCAGG AACCAGTTTA 1782 TGTTTGTGGT TTTGGGAAAA ATTATTTGTG TTGGGGGAAA TGTTGTGGGG GTGGGGTTGA 1842 GTTGGGGGAT ATTTTCTAAT TTTTTGTA CATTTGGAAC AGTGACCAAT AAATGAGACC 1902 CCTTTAAACT GTCAAAAAAAAAAAA A 1933
  • Sequence type nucleic acid
  • Type of row E cDNA to mRNA
  • Organism name ⁇ ⁇
  • CAC TGC AAA AAG CTT GCC CCA GAG TAT GAA GCT GCA GCT ACC AGA TTA 305 His Cys Lys Lys Leu Ala Pro Glu Tyr Glu Ala Ala Ala Thr Arg Leu
  • GGA ATT GTC AGC CAC CTG AAG AAA CAG GCT GGA CCA GCT TCA GTT CCT 497 Gly lie Val Ser His Leu Lys Lys Gin Ala Gly Pro Ala Ser Val Pro
  • GCA AAA GGA GAG AAG TTT GTC ATG CAG GAG GAG TTC TCG CGT GAT GGC 1121 Ala Lys Gly Glu Lys Phe Val Met Gin Glu Glu Phe Ser Arg Asp Gly
  • TAAAGCAGCA GCCAAACATC ATATACTTTG TCAAAGGACT TTTCCACCAG AGATGGGAAA 1658 ACCAATGGGG AGGACTGGGA CCCGTATGGG AATTACTGCC TCTCAGGGCT GAGAGGGCAG 1718 AATGGTTATA ATCTGAGTCC TGTTAAATTT TCTCTATAACT GTTTCTTTGGTC ATGA TGA TGA TGA GATT ATGA AATGTTGTGG GGGTGGGGGGGG AATTGAGTTG GGGGGTTATT TTCTAATTTT TTTTGTACAT 1898 TTGGAACAGT GACAATAAAT GCGCCCCCTT TAAAAAAAAA AAAA 1942

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Abstract

L'invention se rapporte à un gène codant pour un polypeptide de phospholipase C-α (PLC-α) humaine, à un vecteur d'expression contenant ce gène, ainsi qu'à un transformant contenant ce vecteur. Un polypeptide de PLC-α humaine est produit par culture de ce transformant. Ce polypeptide est utile comme anti-inflammatoire et il peut être utilisé pour construire un système de mesure pour l'examen clinique de la cancérisation.
PCT/JP1994/001572 1993-09-24 1994-09-22 PHOSPHOLIPASE C-α HUMAINE ET SEQUENCE D'ADN CODANT POUR CET ENZYME WO1995008624A1 (fr)

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EP94927098A EP0731164A4 (fr) 1993-09-24 1994-09-22 PHOSPHOLIPASE C-alpha HUMAINE ET SEQUENCE D'ADN CODANT POUR CET ENZYME
US08/627,907 US6060302A (en) 1993-09-24 1994-09-22 Human phospholipase C-α and DNA sequence encoding the same

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JP23840293 1993-09-24
JP5/238402 1993-09-24

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996032485A1 (fr) * 1995-04-10 1996-10-17 Incyte Pharmaceuticals, Inc. Homologue de phospholipase c
WO1996040939A2 (fr) * 1995-06-07 1996-12-19 Cadus Pharmaceutical Corporation Expression de phospholipases fonctionnelles de vertebres dans la levure

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040033509A1 (en) 2000-07-18 2004-02-19 Millennium Pharmaceuticals, Inc. Novel 13237, 18480, 2245, 16228, 7677, 26320, 46619, 33166, 16836, 46867, 21617, 55562, 39228, 62088, 46745, 23155, 21657, 42755, 32229, 22325, 46863 and 32252 molecules and uses therefor
ATE433489T1 (de) * 2000-03-31 2009-06-15 Millennium Pharm Inc 16836, ein mitglied der humanen phospholipase c familie und seine verwendungen
WO2001083771A2 (fr) * 2000-04-29 2001-11-08 Merck Patent Gmbh Nouveau element c delta 5 de phospholipase humaine
AU2001261424A1 (en) * 2000-05-11 2001-11-20 Incyte Genomics, Inc. Lipid metabolism enzymes
AU2001291685A1 (en) * 2000-07-31 2002-02-13 Bayer Aktiengesellschaft Regulation of human phosphatidylinositol-specific phospholipase c-like enzyme
US6391606B1 (en) * 2000-09-14 2002-05-21 Pe Corporation Isolated human phospholipase proteins, nucleic acid molecules encoding human phospholipase proteins, and uses thereof
WO2002038773A2 (fr) * 2000-11-08 2002-05-16 Millennium Pharmaceuticals, Inc. 32544, nouvelle phospholipase c humaine et ses applications

Non-Patent Citations (3)

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Title
NATURE, Vol. 334, No. 6179, (1988), BENNETT, C. FROMK et al., "Molecular Cloning and Complete Amino Acid Sequence of Form-I Phosphoinositide Specific Phospholipase C", p. 268-270. *
See also references of EP0731164A4 *
YASUTAKA TAKAGI, "Method for Experimenting Gene Manipulation", 1 July 1981, (KODANSHA SCIENTIFIC), p. 167-168. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996032485A1 (fr) * 1995-04-10 1996-10-17 Incyte Pharmaceuticals, Inc. Homologue de phospholipase c
WO1996040939A2 (fr) * 1995-06-07 1996-12-19 Cadus Pharmaceutical Corporation Expression de phospholipases fonctionnelles de vertebres dans la levure
WO1996040939A3 (fr) * 1995-06-07 1997-01-23 Cadus Pharmaceutical Corp Expression de phospholipases fonctionnelles de vertebres dans la levure

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EP0731164A4 (fr) 2000-11-22
EP1533372A1 (fr) 2005-05-25
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US6060302A (en) 2000-05-09

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